PROJECT SUMMARY/ABSTRACT Burkholderia pseudomallei (Bp), an environmental bacterium capable of infecting humans and animal hosts, is the causative agent of melioidosis, a highly fatal disease endemic in many tropical areas. In addition to its significant public health burden in endemic regions, causing ~89,000 deaths annually, Bp is categorized as a Tier 1 Select Agent of bioterrorism, in large part because inhalation of low doses of Bp can cause rapidly fatal pneumonia. As naturally acquired melioidosis is often difficult to diagnose and requires very lengthy treatment, and as post-exposure prophylaxis is not a practical public health policy for countering an outbreak of pneumonic melioidosis from a bioterrorist attack, a safe and effective pre-exposure vaccine would be highly beneficial in both circumstances. Currently, there are no licensed vaccines against Bp. Hence, a vaccine against Bp would be of great benefit to people living in endemic regions, travelers, and military personnel stationed in these areas, and it would also reduce the risk from an intentional release in a bioterrorist attack. The goal of this application is to develop a safe and highly potent vaccine against Bp, suitable for both biodefense and public health purposes, based on a readily expandable single vector platform vaccine. Specifically, we shall use a novel, safe, yet potent live attenuated vector, LVS ?capB. The LVS ?capB vector, developed in this laboratory, is a further attenuated version (>10,000 fold less virulent in mice) of a multi- deletional Francisella tularensis vaccine (F. tularensis subsp. holarctica Live Vaccine Strain; LVS) already extensively tested in humans; hence, LVS ?capB is anticipated to be exceedingly safe. Moreover, recombinant vaccines comprising this vector have been shown to induce potent humoral and cell-mediated (including both CD4+ and CD8+) immune responses, all likely important to long-lasting potent immunoprotection against Bp. In preliminary studies, homologous boosting with rLVS ?capB vaccines expressing immunoprotective Bp antigens has already demonstrated impressive protective efficacy against respiratory challenge with virulent Bp in sensitive BALB/c mice; protection was superior to the unlicensed attenuated Bp82 vaccine, which is unlikely to be approved because of concerns about reversion to virulence and the possibility of causing chronic disease. To accomplish our goal of developing a safe and highly potent vaccine against Bp, we shall a) construct optimized rLVS ?capB vaccines expressing multiple immunoprotective Bp antigens as fusion proteins; b) evaluate the vaccines systematically in vitro for protein expression, growth kinetics, and stability; c) evaluate the vaccines administered by different routes for safety, dissemination, clearance, and stability in both immune competent and immune deficient mice; d) evaluate protective efficacy against challenge with highly virulent Bp by the respiratory (IN) route, the route of greatest concern in a bioterrorist attack, and by the subcutaneous route, the route principally responsible for natural infection; and e) explore correlates of protective immunity using vaccines of escalating efficacy.